The primary objective of this project is to determine the mechanism by which the microsomal cytochrome P-450-dependent mixed function oxidases catalyze the metabolic activation and detoxification of chemical carcinogens. This enzyme system is present in most human tissues and plays an essential role in the oxygenation reactions which appear to be the first step in metabolism of many carcinogens. Both chloroperoxidase and horseradish peroxidase catalyze the substrates in a manner similar to that of the microsomal system and will be used as model systems for the study of the mechanisms of oxygen activation and substrate oxygenation by microsomal cytochrome P-450. A third model system for these studies will use microsomal preparations and peroxides in place of NADPH and O2. In order to gain a better understanding of the catalytic mechanisms involved in cytochrome P-450-catalyzed dealkylations, the initial experiments are concerned with detailed steady-state and transient-state kinetic studies on peroxide-supported N- and O-dealkylation reactions catalyzed by these model systems. In order to learn more about the substrate binding sites and the interacton of enzyme-bound substrate with the heme iron and/or axial ligands to the heme iron, the interaction between the peroxidases and substrates, inhibitors, and other ligands will be investigated by optical difference spectroscopy and compared with the results obtained from kinetic measurements. The formation of enzyme and/or substrate intermediates during the course of the reactions will be investigated by means of stopped-flow, EPR, and low-temperature spectroscopy.